Effects Of Osteogenic Differentiation In The High Clocuse And Lipid Environment Of HUMSCs And BMSCs

Objective:The multiplication and estrogenic capability of BMSCs and HUMSCs can beevaluated, through observing the effect of the high glucose and high lipid on both of thesetwo stem cells in the inner simulation environment of the dubieties, therefore to settheoretical basis for the recovering treatment of diabetes suffering patients with the help ofbone tissue engineering methods.Methods:I:(1) Isolation and culture of bone marrow Mesenchymal stem cells: After obtainingbone marrow, adopting adherent of Mesenchymal stem cells, using classical densitygradient centrifugation to isolate and culture Bone marrow mesenchymal stemcells.(2)Morphology between primary bone marrow-derived mesenchymal stemcells.(3)Evaluation of bone marrow mesenchymal stem cells：using flow cytometry to testexpression of surface antigen CD29， CD90and CD34, CD45of the BMSCs in thisexperiment.Ⅱ: Multiplication capacity test of BMSCs and HUMSCs as well as the effect of highglucose and lipid on the multiplication capacity. There are four groups in this experiment,collecting the fifth generation BMSCs and HUMSCs and inoculated in96well-plates.Group A: inoculating BMSCS, culture Medium low glucose. Group B: inoculating BMSCs,Culture high glucose and high lipid medium. Group C: inoculating HUMSCs, culturingMedium low glucose. Group D: inoculating HUMSCs, Culturing high glucose and highlipid medium.There are35complex holes in every group, pick5complex holes a day randomly andobserving the cell value by using cck-8way, observing7days continually. Ⅲ: The ability of BMSCs and Umbilical cord mesenchymal stem cells as well as theeffect of high glucose and high lipid its ability. There are four group in this experiment.Group A: BMSCs Blank control group, using the traditional Osteogenic induction medium.Group B: BMSCS experimental group, using the High glucose and high lipid boneinduction medium. Group C: HUMSCs Blank control group, using the traditionalOsteogenic induction medium. Group D: HUMSCs experimental group, using the Highglucose and high lipid bone induction medium.(1) Observing the morphology of cells prior to osteogenic induction and Osteogenicinduction in the3rdday and14thday.(2) Osteogenic induction for7days, staining4groups cells by the Alkalinephosphatase staining kit.(3)Osteogenic induction for21days, staining4groups cells by alizarin red.(4)Osteogenic induction for15days, Type I collagen staining4groups cells by the DBA Staining Kit.(5) By using the Cell activity of alkaline phosphatase colorimetric assay kit,Quantitative detecting the alkaline phosphatase four groups in the7thday,10thday and15thday.(6)After21days osteogenic coparing the expression of Osteocalc in gene through RT-PCR.Results:Ⅰ：(1)About24hours after inoculated, cells attached to the wall, the Fusiform andpolygonal cells can be found3days later, numbers of Long fusiform cells increased9days later and the adjacent colony integrated. The observed cellular(2)Detection of flow cytometry，the experiment Isolated bone marrow mesenchymalstem cells and its surface shows CD29(99.8%),CD90(100%); low expressionCD34(1.4%),CD45(0.2%), Meet the mesenchymal stem cell surface antigen characteristics.Ⅱ： Group C cell proliferation is the fastest, group B and group D are much slower,in platform period, the difference between group A and group C was not statisticallysignificant (P>0.05). group A， C and group B,D have the significances in statistics(P<0.05) Ⅲ：（1） There is an obvious cellular morphology change in the group B and groupD, cells shrinking and disorder.(2)Alkaline phosphates are positive in four groups,however, the positive result are weaker in group B and group D.(3)Results about alizarinred are positive, however, the numbers and of calcium nodule in group B and D are lessand smaller.(4)Collagen I staining results are positive in four groups, however, the stainedpale brown are lighter in group B and D.(5)The activity trend is similar in the four cellgroups, reached the peak point in the10th day and fell obvious in the15th day. Group Band group D have a low expression and the difference with group A and C aresignificant.(6) Osteocalcin gene is expressed by all the four cell groups. There are not anysignificant statistically difference in group A and group(P<0.05).Conclusion:(1) Compared to BMSCs, HUMSCs proliferates faster.(2) In the high glucose andlipid environment, proliferation activity of BMSCs and HUMSCs reduces.(2) In highglucose and lipid environment, although osteogenic differentiation vitality of BMSCs andHUMSCc are damaged, it still has osteogenesis.(3) Under high glucose and lipidenvironment, osteogenic potential of HUMSCs and BMSCs has no significant difference.(4)In the engineering field of bone tissue, BMSCs can be replaced by HUMSCs as seedcells.(5) In the application of bone tissue engineering field to promote fracture healing indiabetic patients HUMSCs has broad prospects.